Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis.
Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.
Kouwen
Thijs R H M
TR
Department of Medical Microbiology, University Medical Center Groningen and University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The Netherlands.
Dubois
Jean-Yves F
JY
Freudl
Roland
R
Quax
Wim J
WJ
van Dijl
Jan Maarten
JM
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D010084:Oxidation-Reduction; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D011490:Protein Disulfide Reductase (Glutathione); D000469:Alkaline Phosphatase; C114089:phoA protein, E coli